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Learn why ChIP is such a useful laboratory technique, how to optimize your experiment protocol and discuss key troubleshooting tips with our Epigenetic specialist.
David Grotsky received his PhD in Molecular Genetics and Genomics from Washington University in St. Louis. His PhD thesis focused on studying the regulation of DNA repair factors and how they impact genomic stability and proliferation in tumor cells.
David is currently the Epigenetics Specialist on Abcam’s Scientific Support Team.
Thanks very much Jessica. Hi everyone, I’m Dave and I’d like to thank you all for joining me here today.
I’d like to start by just giving an overview of what I’ll be discussing in this webinar. I’ll start with an introduction to chromatin structure and histone modifications, followed by a brief summary of what ChIP is used for and why it’s such a powerful tool. Then I’ll talk about the ChIP protocol and go into specifics about optimizing your ChIP protocol and troubleshooting problems.
Let’s begin by talking about chromatin. In the human genome there are around 3.2 billion DNA base pairs, which if unraveled would stretch to around 5 feet of DNA in every cell. To remedy this, DNA is tightly compacted into a higher order structure called chromatin so that the DNA can fit inside a cell’s nucleus. Chromatin, as you’ll see is also an important factor in controlling gene expression. The most basic unit of chromatin is the nucleosome, which consists of DNA wound around a core of eight histone proteins. The nucleosome core consists of approximately 147 base pairs of DNA wrapped around the histone octamer consisting of 2 copies each of histones H2A, H2B, H3, and H4. Histone H1 sits on top of this structure to keep the wrapped DNA in place. This beads on a string structure is euchromatin. When multiple nucleosomes condense even further to wrap into the 30nm fiber, this is termed heterochromatin. Finally there is even higher level packaging to form metaphase chromosomes during mitosis.
The histones I mentioned earlier can undergo many post translational modifications that affect how they interact with DNA, some of which are shown here. Specific modifications have different effects on gene transcription, creating a sort of histone code, an idea that was popularized in the early 2000s. Histones H3 and H4 are the most commonly modified histones and have long N terminal tails protruding from the nucleosome whose residues are targeted for acetylation, methylation, phosphorylation, deamination, and ubiquitylation. Acetylation is generally associated with gene activation, while different and specific methylation modifications are involved with either gene activation or repression by relaxing or condensing chromatin to allow transcription factors to bind to the promoter region of a gene. Being able to investigate exactly where these histone modifications are present and where transcription factors are binding in the genome was made possible with the advent of chromatin immunoprecipitation.
ChIP is an experiment that allows you to measure the direct interaction of DNA and proteins in the cell in a given cellular state, condition or time point. As I said, it’s useful for determining where histone modifications are located in the genome and where transcription factors bind to promoters and other DNA binding sites. In order to perform ChIP you need to use antibodies to bind the proteins that are bound to DNA, and here I’m showing an example of a ChIP experiment using an antibody targeting histone H3 that’s trimethylated at lysine 4. This histone modification is associated with gene activation, so as we can see there is enrichment for this modification at these 3 active genes but the protein is not present at the 3 inactive genes. Here the readout for the ChIP was performed using q-PCR for one gene, or locus, at a time, but you can also perform ChIP on a genome wide scale with ChIP on chip or more commonly ChIP followed by sequencing the pulled down DNA.
Here we can see how ChIP is such a powerful tool that can produce so much data. I’m showing you the epigenetic profile of an active gene, this data was compiled by performing ChIP seq at thousands of known active genes to see what histone modifications are present. When transcription is happening, histone modifiers introduce activating histone modifications as the polymerase travels along the DNA. The promoter region has a very well defined chromatin signature, there’s a clear nucleosome depleted region where the polymerase binds and large increases in activating H3K4 methylation and acetylation. As transcription continues along the gene, modifiers introduce new modifications to allow the polymerase to move along the DNA.
These genome wide ChIP studies have revealed large scale epigenetic profiles at many different genomic elements. Here I’m showing a dashboard of histone modifications, there are specific modifications at promoters depending on whether they are active, poised or inactive; specific modifications within gene bodies; at enhancers; and modification that are marks of large scale repression and heterochromain. As I showed earlier, H3K4 methylation and acetylation are common at active promoters and enhancers while H3K9 di and trimethylation are common at inactive genes.
Now that we’ve seen an example of the kind of data we can gather using ChIP, I’d like to briefly go over the protocol. There are two kinds of ChIP – cross linking chip and native chip, and I will go into much greater detail about the two in a bit. In cross linking ChIP, DNA is crosslinked to proteins using formaldehyde, cells are lysed, and the DNA is then fragmented by sonication into random fragments of 200-1000 base pairs. In native ChIP, the DNA and proteins are not cross linked and micrococcal nuclease is used to digest the DNA, which can produce DNA fragments at the level of a single nucleosome, or around 150 to 175 base pairs. Next, a specific antibody of interest is added to bind to the protein DNA complex which is then immunoprecipitated, pulling down only DNA fragments that are directly bound to the target protein. Crosslinks are then reversed if you’re doing cross linking chip and the DNA can be purified for later analysis by PCR, hybridization to a microarray, or sequencing. The abundance of DNA pulled down can be compared to the input, or starting, chromatin amount.
As I just mentioned, there is native ChIP and crosslinking ChIP and each has its own advantages and disadvantages. With native ChIP you typically get more efficient precipitation of the DNA and protein; as well as greater specificity because the binding is more predictable. You also have higher resolution, as low as a single nucleosome compared to 200 to 1000 base pair fragments you get with sonication. The disadvantages of Native ChIP include the fact that it’s only for histone proteins because the DNA is tightly interacting with these proteins. If you want to do chip of transcription factors, since their binding to DNA is not strong you’ll have to cross link to ensure the protein DNA interaction remains intact. Another disadvantage is that since the nuclease digestion is selective, the digestion may bias input chromatin; and there are also chances of chromatin rearrangement. And of course if you use too much nuclease you could over-digest the chromatin.
Cross linking ChIP on the other hand is much more flexible and can be performed with histone and non-histone proteins on all cell types, tissues and organisms. In addition to DNA-protein analysis you can also analyze RNA-protein and higher order protein-protein-DNA interactions; and there is a reduced chance of chromatin rearrangements. There are of course several disadvantages of cross linking chip including the facts that over-fixation can prevent effective sonication; enzymatic digestion won’t be possible after formaldehyde treatment; formaldehyde can alter the binding of the antigen; and it has a lower resolution than native chip since you can’t use micrococcal nuclease for digestion. Hopefully now you’ll be able to determine if native chip or crosslinking chip is more appropriate for your experiment.
Now I’d like to talk about optimizing your protocol, starting with how to choose the right antibody. Most antibodies will not work for ChIP, so a good place to start is to look for antibodies that have been fully characterized for chip. You should also look for specificity testing such as peptide array data, here’s that H3K4 trimethyl antibody I showed earlier and we can see the antibody is binding strongly to the K4 trimethyl peptide, with minimal or no binding to these other peptides tested in the array indicating the antibody is specific for the tri methyl K4 modification.
If an antibody is not tested for ChIP but you’re interested in trying it out, look for antibodies that have been tested for IP, IHC, or ICC. These applications recognize the protein’s native conformation, similarly to ChIP, rather than a western blot only antibody that might only recognize a protein in its denatured form. If you’d like to test one of Abcam’s antibodies in ChIP, you can use our Abtrial testing program, which I’ll discuss more later.
Finally, with regards to clonality, polyclonal and monoclonal antibodies both have advantages and disadvantages. Polyclonal antibodies recognize several epitopes which is great for cross linking chip where epitopes might become masked, but they do have some batch to batch variation. Monoclonal antibodies recognize only a single epitope, so if the epitope is masked the antibody won’t bind, but there is minimal batch to batch variation. We have many polyclonal antibodies for ChIP and are currently developing highly specific rabbit monoclonal antibodies to be used in ChIP.
Probably the most important step in optimizing your chip protocol is beginning with a really good chromatin template and the correct fragment size. For cross linking chip this involves finding the appropriate cross linking and shearing time, and for native chip this involves finding the appropriate amount of enzyme and digestion time. You should perform a time course to determine the right amount of time to crosslink, which could be anywhere from 2 to 30 minutes, but 10 to 15 minutes is usually sufficient. Getting the right fragment size is of utmost importance, you must try different sonication times and then purify DNA to run in a gel to figure out what sonication time will get you the appropriate size. You typically want fragment sizes of 200 to 100 base pairs. For native chip you can get fragments around 175 base pairs.
Next you should determine the proper amount of antibody to use for your ChIP. Typically around 4 ug of antibody per 25 ug of chromatin is appropriate, but the antibody should be titered to improve the signal to noise ratio. The composition of your wash buffers is important, you should determine the appropriate amount of sodium and lithium chloride to provide the appropriate stringency. This could be from 250 to 500 milimolar. Having a wash buffer that isn’t stringent enough will lead to high background, while a buffer that is too harsh will destroy specific antibody interactions.
Since chip is difficult and time consuming to perform it’s very important to have the right controls to determine if the experiment worked properly. These include a negative IP control, which would be an isotype igg control or beads only; positive and negative control loci for qPCR, which would be a region or gene where you know a protein should be bound and shouldn’t be bound; and a non-template control for qPCR to make sure there’s no contamination in your PCR.
Positive controls are extremely important. If you’re looking at an active locus you can use antibodies that recognize H3k4 trimethyl or H3k9 acetyl for instance, and if you’re looking at a repressed locus you can use antibodies that recognize h3k9 trimethyl or h3k27 trimethyl. You can also you a histone H3 antibody which should present everywhere. Here you can see the presence of H3K4 trimethyl at active loci and H3K9 trimethyl at heterochromatin.
Now on to troubleshooting. What if you have high background in your non-specific antibody control? So this would be either your isotype igg control or beads only tubes. There could non specific binding to the beads, so I always recommend including a pre clearing step. You could have contaminated wash buffers so its good practice to always use fresh buffers or replace your buffers if you’re getting high background. And some beads inherently have high background, so it’s good to try different beads and different blocking strategies to see which provide the lowest background in the non specific control.
What if you have low resolution with high background across large regions? This is most likely due to the DNA fragment size being too large. Fragments should definitely not be larger than 1.5 kilobases and ideally should be between 200-1000 bp. And keep in mind if you’re able to do native chip you can get the best resolution of single nucleosomes.
What if you’re getting low or no signal? Well, lots of things could be wrong. The DNA fragment size could be too small, make sure you run your fragments on a gel to see if they’re the correct size. If you’re performing cross linking chip the cells may have been crosslinked for too long which can reduce the availability of epitopes, so be sure to perform a time course and quench with glycine. It could be that you don’t have enough starting material, ChIP generally requires a large amount of input, around 25 ug of chromatin per ip or 3-4 million cells per IP. You could also not have used enough antibody, 3-5 ug is usually sufficient but up to 10 ug might be required if there’s low or no signal.
You could be eliminating the specific antibody binding if your wash buffers are too stringent, so don’t use more than 500 milimolar of sodium or lithium chloride. Make sure your cells are sufficiently lysed, something like a RIPA buffer should work well. Keep in mind that your result might be real if there’s no antibody enrichment at the region of interest, be sure to include a positive control antibody and locus to ensure the ChIP is working.
Make sure you know when to use native vs cross linking chip. Cross linking chip is more suitable when analyzing proteins that have weaker DNA affinity so crosslinking may be required to keep proteins associated with DNA; and native chip can really only be performed if you’re looking at histones. If you’re using a monoclonal antibody it may not be suitable for cross linking chip due to epitope masking, so try an antibody that’s tested for chip or a polyclonal antibody. And make sure you’re using the correct affinity beads, so make sure the antibody species and immunoglobulin bind to the chosen beads or use a protein A/G mix.
What about problems with the PCR amplification? If you’re getting high signal in all samples after PCR including the non-template control, your pcr solutions may be contaminated so you should use new solutions. And if you’re getting no amplification in your samples be sure to include a standard or input DNA to confirm the primers are working.
I’d like to turn now to the resources Abcam provides to help scientists with their research. I mentioned the Abtrial program earlier, this is a program where you can use our products in an untested application or species without financial risk. This is great program if you’d like to try out one of our untested antibodies in ChIP. If you send us your results in an abreview, whether they’re positive or negative, we’ll send you a discount code for the full value of the product that can be used on a future order. If you’d like to enroll in this program, you can visit abcam.com/abtrial or contact our scientific support department. I’ll show our global scientific support contact information a little bit later on.
We also have our abreview program, these are user reviews from customers like yourselves that show whether or not a product works and exactly what they did to get the product to work. You can submit a review for any of our products on a scale of 1 to 5 stars and we publish all of them, good and bad. Here you can see this H3k9 acetyl antibody has 32 reviews, and if you click on this link here
You’re able to filter the reviews by application, species, and by rating, and see the specific protocol information and an image for each review.
We also gather references for our products, when we find them or hear about them from customers we make them easy to access on every datasheet by clicking this link. You can see this antibody has 88 references and when you click here
It gives you a list of the publications as well as the application and species that the antibody was used for in.
I’m very excited to tell you about our first conference in Australia, on april 21 we’re hosting “mechanisms and mysteries in epigenetics” in Sydney. The conference chairs are Catherine Suter and John Mattick and confirmed speakers include John Stam from university of washington, Oded Rechavi from Tel Aviv university, and Stirling Churchman from harvard. For more information please visit abcam.com/epigeneticsAU.
We also have a ton of epigenetics resources on our website, just visit abcam.com/epigenetics to see articles, events, products, protocols, and our webinar library.
Finally, we’re here to help! If you ever have questions or problems with our products, don’t hesitate to contact us by email or phone, we offer support around the globe and in many different languages. Also keep in mind that all of our products are covered by our Abpromise guarantee, if you’re using a product in a tested application and species and it’s not working as it says it should on the data sheet just let us know and we’ll help you get the product working or give you a replacement or refund. To contact us just click the contact us link at the top of our website and I’ll be showing our contact information while I answer some questions in a sec. Thank you very much for joining me, I’ll now turn this back over to Jessica before I answer your questions.